Agrobacterium Tumefaciens-Mediated Transformation of Tobacco (Nicotiana Tabacum L.) Leaf Disks: Evaluation of the Co-Cultivation Conditions to Increase Beta-Glucuronidase Gene Activity
Agrobacterium tumefaciens-mediated transformation is generally used for genetic transformation of higher plants. Several experimental factors important for the increase of beta-glucuronidase (GUS) reporter gene activity were evaluated in this study using leaf disks of tobacco (Nicotiana tabacum L. cv. Xanthi). We found that co-cultivation temperature at 20C is the most critical factor to obtain the reproducible enhancement of GUS activity. pCAMBIA 1305.01 resulted in higher GUS activity than the other two pCAMBIA vectors 1301 and 1305.02.
The highest GUS activity and transformation efficiency were achieved under the following experimental conditions: Agrobacterium tumefaciens strain LBA4404 containing pCAMBIA1305.01 was grown overnight at 28oC in liquid Agrobacterium media, and the concentration was adjusted to 3x107 cells/mL (0.3 A600 units/mL). Tobacco leaf disks were inoculated with bacteria under 50 mm Hg vacuum infiltration for 20 min in the presence of 0.001% (w/v) Silwet L-77. Leaf disks were co-cultivated for four days under constant light at 20C in MS shoot media containing 200 uM acetosyringone without antibiotics. Leaf disks were then transferred to MS shoot selection media containing 50 mg/L hygromycin and 500 mg/L carbenicillin, and grown for an additional 14 days under constant light at 25C. Beta-Glucuronidase (GUS) activity was measured at the end of the growth period by quantitative GUS assay and GUS histochemical staining.
Advisor:Norimoto Murai; Marc Alan Cohn; John Dyer
School:Louisiana State University in Shreveport
School Location:USA - Louisiana
Source Type:Master's Thesis
Keywords:plant pathology crop physiology
Date of Publication:07/10/2006